When H. pylori in the stomach was linked with gastric cancer, and its eradication associated with regression, a paradigm-shifting model of carcinogenesis was created. Many bacterial species normally reside in various regions of the oropharyngeal mucosa, and may fluctuate depending on the host's age and dietary consumption. Interestingly, the incidence of head and neck squamous cell carcinoma (HNSCC) has been shown to correlate with dental hygiene, which may suggest the relevance of bacteria in the pathogenesis of this particular malignancy. Some bacteria can result in inflammation, which can cause increased methylation. Using genomic technology, we have demonstrated a possible link between HNSCC and specific bacterial populations based on their metagenomic profiles. Notably, we have proof-of-principle association between the presence of particular bacterial subpopulations per metabiomic profiling and MDR1 methylation status in HNSCC. We therefore hypothesize that specific bacterial subpopulations, as revealed by metagenomic profiling, can trigger targeted methylation events which then initiate HNSCC genesis and/or progression. We plan to address this hypothesis via 3 broad aims: (1) To utilize most recent high throughput sequencing technologies to identify the bacterial subpopulations in the oropharyngeal mucosa associated with HNSCC tumor initiation, progression, and/or prevention, with the innovative sub-aim to explore if specific microbiomic profiles are part of the field effect seen in HNSCC;(2) To interrogate the specific microbial "consortia" influencing inflammation with consequent epigenetic alterations of target tumor suppressor genes in HNSCC;(3) To functionally validate the involvement of MAD2 and/or MDR1 in HNSCC tumor suppression. The resulting microbial populations as reflected by metagenomic profiles will be interrogated for correlation with exposure and clinical information and targeted methylation. Finally, we will functionally validate whether the targeted methylation is relevant in HNSCC tumorigenesis or a random methylation effect in response to inflammation. To address this, we will overexpress or knock-down MDR1 in HNSCC cell lines and examine any effect on growth, apoptosis, cell morphology, or differentiation. This study promises to reveal the relevance of specific head and neck bacterial flora to cancer initiation and progression. Interesting future directions include clinical screening for the presence of specific bacterial indicators in order to provide early cancer or pre-cancer detection and perhaps prevention, based on probiotics or antibiotics. With metabiomic profiles capable of distinguishing samples based on prior radiation exposure or chemotherapy, we will implement targeted screening and/or demethylation treatment for patients with refractory disease. This type of data will also point to larger roles for early detection, awareness education, and prevention from hygienists and nursing. PUBLIC HEALTH RELEVANCE: Over 500,000 new cases of head and neck cancers (HNSCC) are diagnosed worldwide annually, with more than 11,000 individuals/year succumbing to this aggressive malignancy in the United States alone;despite huge strides in research achievements in this topic, overall outcome has remained unchanged for over 3 decades for the majority of patients with advanced stages of this disease. Patients who receive aggressive surgery to remove the cancer have significant disfigurement and diminished quality of life, with impaired ability to swallow, smell, and hear. If successful, then our current proposal promises a new understanding of the role of specific subpopulations of bacteria in the mouth leading to inflammation which shuts off cancer-suppressing genes, thus leading to HNSCC. If true, then we can take advantage of our findings for the earliest diagnosis and even prevention with probiotic therapy. Importantly, even for advanced HNSCC or that which has spread, our findings will help point to antibiotics and agents that can turn back on the cancer-suppressing genes to revolutionize treatment.